Protein restriction slows the development and progression of Alzheimer's disease in mice

Abstract Over the last decade, it has become evident that dietary protein is a critical regulator of metabolic health and aging. Low protein diets are associated with healthy aging in humans, and we and others have shown that dietary protein restriction (PR) extends the lifespan and healthspan of mice. Here, we examined the effect of PR on metabolic health and the development and progression of Alzheimer’s disease (AD) in the 3xTg mouse model of AD. We found that PR has metabolic benefits for 3xTg mice and non-transgenic controls of both sexes, promoting leanness and glycemic control in 3xTg mice. We found that PR induces sex-specific alterations in circulating metabolites and in the brain lipidome, downregulating sphingolipid subclasses including ceramides, glucosylceramides, and sphingomyelins in 3xTg females. Consumption of a PR diet starting at 6 months of age reduced AD pathology in conjunction with reduced mTORC1 activity, increased autophagy, and had cognitive benefits for 3xTg mice. Finally, PR improved the survival of 3xTg mice. Our results demonstrate that PR slows the progression of AD at molecular and pathological levels, preserves cognition in this mouse model of AD, and suggests that PR or pharmaceutical interventions that mimic the effects of this diet may hold promise as a treatment for AD.


Introduction
The global population is aging, resulting in a growing burden on health care systems around the world.Aging is the most profound risk factor for Alzheimer's disease (AD), which currently affects over 5 million Americans; by 2050, over 13 million Americans are expected to have AD, with a total health care cost of over $1 trillion, placing an immense burden on healthcare resources 1 .Obesity and diabetes, now widespread in the population, further increase the risk of AD; while the exact relationship is unclear, the risk of AD is increased by type 2 diabetes, and glucose metabolism is impaired in individuals with AD (reviewed in 2 ).4][5] .Thus, identifying new and effective interventions that can delay or prevent AD are of great importance.
Caloric restriction (CR) is the gold standard for geroprotective interventions, extending lifespan and healthspan in diverse species 6 .CR slows or prevents the development of AD in multiple mouse models [7][8][9][10] as well as squirrel monkeys 11 .In humans, the effect of CR on AD is unknown, but short-term CR can improve memory 12 .The powerful benefits of CR on AD more broadly suggests that geroprotective interventions may be able to slow or prevent AD.
While CR is difficult for most people to adhere, diets that alter the level of specific macronutrients without restricting calories may be more tolerable 13 .5][16][17][18][19][20] Moreover a study published in 2013 by Parrella et al.   found that repeated cycles of a protein-free diet improved cognition in a mouse model of Alzheimer's disease.This highlights the potential benefits of protein restriction not only on metabolic health but also on cognitive function 21 .In rodents, PR has been shown to promote metabolic health and even extend lifespan [22][23][24][25][26][27] .
Although the mechanisms by which PR promotes metabolic health and lifespan are not fully understood, amino acids are strong agonists of the mechanistic Target Of Rapamycin Complex 1 (mTORC1), a highly conserved nutrient sensing protein kinase that acts as the central regulator of many cellular processes 28 .Genetic or pharmaceutical inhibition of mTORC1 extends lifespan in diverse species [29][30][31][32][33] .As PR necessarily involves reduced dietary consumption of all amino acids, PR inhibits mTORC1 activity in multiple tissues in mice 24,34 .mTOR signaling is strongly linked to AD. Increased levels of phospho-mTOR and two of its downstream targets, p70S6K and the eukaryotic translation factor 4E (eIF4E) have been visualized in the brains of patients with AD. [35][36][37][38] .While phosphorylation of these substrates acts to promote macromolecule synthesis, mTORC1 activity acts as a key negative break on autophagy, via the phosphorylation of ULK1 39,40 ; consequently, increased mTORC1 activity accelerates the production of Aβ and accumulation of tau 38,41 .Furthermore, treatment with rapamycin, a drug which inhibits mTORC1 activity, slows or prevents AD in multiple mouse models of AD, and genetic depletion of S6K1, a substrate and effector of mTORC1, is sufficient to improve memory and reduce AD pathology in 3xTg mice 38,[42][43][44] .
As PR decreases mTORC1 signaling in multiple tissues, and inhibition of mTORC1 signaling promotes healthy aging and blunts AD in mouse models, we hypothesized that PR might slow or prevent the progress of AD.Although the concept that dietary composition may impact the development of AD has not been well-explored, repeated cycles of a protein free diet improves memory in a mouse model of AD 21,45 .Similarly, restriction of the branched-chain amino acids leucine, isoleucine, and valine -which are strong agonists of mTORC1 activity and are necessarily reduced in PR diets -was recently shown to improve novel object recognition in a mouse model of AD, although it did not decrease tau phosphorylation 46 .
We tested the hypothesis that PR has beneficial effects on the progression of AD pathology and cognitive loss in the 3xTg mouse model of AD.The 3xTg mouse model, expresses familial human isoforms of APP (APPSwe), Tau(tauP301L), and Presenilin (PS1M146V), and exhibits both Aβ and tau pathology 47 , as well as cognitive deficits 48,49 .Further, the 3xTg model has been used to examine the effect of many different geroprotective interventions on AD 44 .Here, we fed 3xTg mice as well as non-transgenic (NTg) controls either a Control diet (21% protein) or a PR diet (7% protein) starting at 6 months of age, which is after the age at which 3xTg mice develop cognitive deficits and have intracellular Aβ immunoreactivity in parts of the hippocampus and cortex 50 .We assessed the effect of PR on metabolic health, AD neuropathology, cognition, and survival of 3xTg and NTg control mice.
We found that a PR diet has beneficial metabolic effects in both 3xTg and NTg mice of both sexes, reducing adiposity, normalizing glucose metabolism in glucose intolerance and insulin sensitivity strongly in 3xTg females.PR induces significant sex-dependent alterations in circulating metabolites and brain sphingolipids in 3xTg mice.Critically, we find that PR improves multiple aspects of AD pathology, reducing the density of Aβ plaques as well as decreasing Tau phosphorylation in both males and females.These positive effects on AD pathology are associated with reduced mTORC1 activity and activation of autophagy.Finally, we show that PR improves the cognitive performance of both male and female 3xTg mice and increases the survival of 3xTg mice.Our findings suggest that PR, or drugs that mimic the effects of PR, may be a novel way to slow or even prevent the progression of Alzheimer's disease.

Dietary protein restriction after disease onset reduces bodyweight and improves metabolic health in 3xTg mice.
We randomized six-month-old male and female 3xTg mice and non-transgenic (NTg) control mice to one of two diet groups: a Control diet (21% of calories from protein) or a Protein Restricted (PR) diet (7% of calories from protein), which we have utilized in a previous study 27 .
Briefly, these two diets are isocaloric, with the calories from fat kept constant, and the level of carbohydrates increased in the PR diet to replace calories from protein (Table S1).We followed the mice longitudinally, tracking their body weight monthly and determining their body composition at the beginning and end of the experiment after 9 months on diet.The experimental design is summarized in (Fig. 1A).
Both NTg and 3xTg female mice fed a PR diet maintained their body weight over the course of the nine-month study, while mice of both genotypes fed the Control diet gained weight (Fig. 1B).PR-fed female mice had significantly reduced accretion of fat mass during the ninemonth study, while lean mass in PR-fed mice similarly remained constant; by the end of the experiment, we observed an overall effect of diet on both lean and fat mass (Figs.1C-D).Thus, by the completion of the study, PR-fed females of both genotypes had reduced adiposity compared to Control-fed females (Fig. 1E).These changes in body weight and body composition were not the result of reduced caloric intake; rather, as we and others have previously observed in other mouse strains 27 , female mice fed a PR diet consumed more food than Control-fed females (Fig. 1F).
The result of PR feeding on the weight and body composition of males was similar to the response of females, but complicated by the fact that the 3xTg males began the study weighing less than NTg males, primarily due to significantly lower initial fat mass (Figs.1G-I).Both genotypes of male mice consuming the PR diet either gained less fat mass or lost fat mass, during the study and ended the study significantly leaner than Control-fed males of the same genotype (Fig. 1H-J).Surprisingly, and unlike in females, we did not observe a significant effect of PR on food consumption; instead, 3xTg males ate more than NTg males regardless of diet (Fig. 1K).
Since the mice on PR diets gained less weight than Control-fed mice despite similar or increased food consumption, we examined the energy balance of all groups using metabolic chambers.We assessed substrate utilization by examining the respiratory exchange ratio (RER), which is calculated using the ratio of O2 consumed and CO2 produced; the RER approaches 1.0 when carbohydrates are being primarily utilized for energy production and approaches 0.7 when lipids are the predominant energy sources 51 .We found no overall effect of diet or genotype on RER in females, although there was a non-significant (p=0.095)effect of genotype during the light cycle, with 3xTg mice having a lower RER (Fig. 2A).We expected to find that PR increases energy expenditure, and we observed this effect in NTg females; however, there was no effect of PR on energy expenditure in 3xTg females (Fig. 2B).There was an overall effect of genotype on activity, with 3xTg mice having greater activity; and there was a significant interaction of genotype and diet during the light cycle, with NTg females and not 3xTg females increasing their activity in response to PR (Fig. 2C).The results of PR in males was somewhat different from the effect in females, as PR increased RER in 3xTg males during the light cycle, and PR increased energy expenditure in both NTg and 3xTg males (Figs.2D-E).PR-fed males of both genotypes trended towards increased activity, with 3xTg mice on PR having the greatest activity (Fig. 2F).
3xTg mice have previously shown to have impaired glucose tolerance which worsens with age 52 .We assessed the effect of PR on glycemic control, performing a series of glucose (GTT), insulin (ITT) and pyruvate (PTT) tolerance tests starting at 9 months of age, after the mice had been on the indicated diet for three months (Fig. 3).In line with the reports of impaired glucose handling in 3xTg mice, we found that Control-fed female 3xTg mice were glucose intolerant at 9 months of age relevant to NTg mice of the same age; however, this was not the case in males, where the area under the curve between Control-fed mice 3xTg and NTg mice was indistinguishable (Figs.3A and 3E).There was an overall positive effect of PR on glucose tolerance in both males and females, which reached statistical significance in 3xTg mice of both sexes (Figs.3A and 3E).
Blood glucose in Control-fed female 3xTg mice was relatively resistant to I.P. administration of insulin, an effect that was reversed by PR feeding (Fig. 3B and 3F).In contrast, PR increased the insulin sensitivity of both NTg and 3xTg males (Fig. 3F).PR improved pyruvate tolerance in both male and female 3xTg mice, suggesting increased suppression of hepatic gluconeogenesis by PR (Figs. 3C and 3G).In summary, long-term intervention of PR improved the metabolic health of both NTg and 3xTg mice of both sexes, with PR generally rescuing the glycemic control abnormalities of 3xTg mice, particularly the females (Fig. 3D and 3H).

Sex-dependent alterations in metabolism in response to PR
To gain additional insight into the effects of PR, we pursued a targeted metabolomics approach, identifying a total of 38 metabolites in the plasma of NTg and 3xTg mice of both sexes (Table S2-S3).Principal Component Analysis (PCA) showed that PR had distinct effects in male and female 3xTg mice; a similar pattern was observed in NTg control mice (Fig. 4A, Fig. S1A).
Figure 4B presents a heatmap illustrating the log2 fold changes from the control diet in the top 25 altered metabolites for both genders.Among females, significant downregulation was observed in plasma metabolites UTP, GTP, and PEP, while Histidine, Citrate, and Ornithine exhibited a significant upregulation because of PR diet feeding (Fig. 4C).In contrast, males exhibited decreased levels of plasma metabolites proline, arginine, and guanosine, accompanied by a significant elevation in uridine concentration in serum (Fig. 4D).These results were reflective of the sex-specific impact on PR on the levels of plasma metabolites in 3xTg mice (Figs.4B-D, Fig.

S1B, Table S4).
We utilized Metaboanalyst to perform KEGG pathway enrichment analysis on the differentially altered metabolites to identify pathways engaged by PR in males and females (Table S5).We found that PR engages distinct pathways in 3xTg males and females; the only pathway we identified as altered in both sexes was "Cysteine and methionine metabolism", which was downregulated in males and upregulated in females (Fig. 4E).Looking at individual metabolites, we found that this was reflective of differential regulation of plasma methionine and its catabolites in each sex (Fig. 4F).Specifically, the metabolites L-Methionine and Glutathione were significantly downregulated in females but not in males, while L-Cystathionine was significantly upregulated in females but not in males.Interestingly, and in contrast to the clear sex-specific effects of PR in 3xTg mice, PR engaged several common pathways in NTg males and females, including Glycolysis/Gluconeogenesis, Folate biosynthesis, and Purine metabolism (Fig. S1C, Table S5)."Cysteine and methionine metabolism" was upregulated in both male and female NTg mice in response to PR, reflecting in part upregulation of L-methionine levels by PR in both sexes (Fig. S1D).
Dysregulated lipid metabolism and altered lipid composition have been implicated in the pathology of AD 53 , and we therefore decided to look directly at the effects of PR on the brain.We performed untargeted lipidomic analysis of the hippocampus and cortex of 3xTg mice that were fed either a Control diet or a PR diet from six to twelve months of age (Tables S6-S7).PCA for both male and female 3xTg mice demonstrated a distinct segregation of lipid classes based on brain regions, but no separation was found based on diet (Figs.S2A-2B).
We next generated a heat map utilizing LION ontology and identified the most significantly modified lipid classes in both the hippocampus and cortex of 3xTg mice (Figs.S2C-2D).We observed that several lipid classes and sub-classes were altered by PR.In particular, the lysophosphatidylethanolamines (LPE), Ether PE lipids, and sphingolipids were altered by PR in both the sexes especially in the cortex.(Figs.S3A-C, E-G).The LPE lipids have been associated with inflammation and oxidative stress and are considered to be involved in the development and progression of AD 54 , while Ether PEs have been suggested to have neuroprotective properties that could help to diminish some of the damaging effects of AD pathology 55 .Pathway enrichment analysis of the complete lipidomic dataset from cortex is depicted in Figs.S3D and 3H (Tables S8-S9).One of the common pathways shared between male and females was increased glycerophospholipids, which are significantly decreased in the hippocampus and cortex of AD patients 56 .Some of the lipid classes altered by PR in a sex-specific manner included sphingolipids, glycerophospholipids and lysoglycerophospholipids.
Over the past two decades, significant focus has been directed towards brain sphingolipids in relation to AD 57,58 .Sphingolipids have been implicated in crucial processes, such as Aβ processing and aggregation, and they also mediate a cytotoxic signal initiated by Aβ 59,60 .
In the 5xFAD mice model of AD, it has been shown that decreasing levels of specific sphingolipids can ameliorate cognitive decline as well as plaque aggregation 61 .Since these are key contributors to the development and progression of AD, we conducted a targeted analysis of sphingolipids in the brains of male and female NTg and 3xTg mice fed either a Control or PR diet (Fig. 5).A heat map showing the relative abundance of sphingolipids in the whole brain of both sexes of NTg and 3xTg mice fed either a Control or PR diet is depicted in Figs.5A and 5E.
Interestingly, we observed an overall decrease in various sphingolipids in the brains of both male and female 3xTg mice.Specifically, in 3xTg females, the PR diet led to significant decreases in subclasses of glucosylceramides, ceramides, and sphingomyelins (Figs.5A-5D).; and in contrast, PR significantly downregulated glucosylceramides and ceramides in 3xTg males.(Figs.5E-5H).

Protein restriction improves AD neuropathology in 3xTg mice.
We next assessed whether PR rescues the progression of AD neuropathology by evaluating several pathological hallmarks of AD, including phosphorylation of tau, amyloid beta (Aβ) plaque deposition, and gliosis.In our study we evaluated AD pathology in 3xTg mice at 15 months of age, after 9 months of consuming Control or PR diets.We observed significantly lower plaque density in the hippocampus of PR-fed 3xTg females than in Control-fed 3xTg females (Fig 6A-B).PR feeding also reduced the soluble fractions of Aβ40, consistent with the effects of the diet on plaque load (Fig. 6C).To investigate the effect of PR on tau phosphorylation, we performed western blotting and found that PR-fed 3xTg female mice reduced tau phosphorylation compared to control-fed 3xTg females (Fig. 6D).
Finally, neuroinflammation is a key pathological feature of AD, and we assessed activation of astrocytes and microglia via immunofluorescence.We found that Control-fed female 3xTg mice had more astrocytes and microglia, as assessed via staining for via GFAP and Iba1, respectively, than PR-fed 3xTg females (Fig. 6E).
In contrast, we found that PR did not significantly reduce Aβ plaque density or soluble Aβ40 in male 3xTg-AD mice, although there was general trend towards reduced levels of both plaques and soluble Aβ40 (Fig. 6F-H).There was a statistically significant decrease in tau phosphorylation in PR-fed 3xTg males (Fig. 6I).However, we observed no effect of PR-feeding on the activation of astrocytes and microglia in the brains of 3xTg males (Fig. 6J).Taken together, these results strongly indicate the beneficial effects of PR on AD pathology in 3xTg mice, particularly in females (Fig. 6K).

Protein restriction reduces mTORC1 hyperactivation and p62 expression in 3xTg mice.
mTOR hyperactivation plays a crucial role in the pathology of AD 38,62, ,63 .As PR can reduce mTORC1 activity, this led us to hypothesize that inhibition of mTORC1 signaling in the brain mediates the benefits of PR on AD pathology.We conducted western blotting to examine the effect of PR on phosphorylation of the mTORC1 substrates T389 S6K1 and T37/S46 4E-BP1, which significantly activated mTORC1 in NTg and 3xTg mice of both sexes.Consistent with a role for increased mTORC1 activity in the etiology of AD, we observed increased phosphorylation of T389 S6K1 and T37/S46 4E-BP1 in the brains of control-fed 3xTg female and male mice relative to control-fed NTg mice (Figs.7A-C, S4A-C).PR significantly reduced the phosphorylation of both T389 S6K1 and T37/S46 4E-BP1 in both female and male 3xTg mice (Figs.7A-C, S4A-C).
The dysregulation of autophagy is a key pathophysiological feature of AD; moreover, the hyperactivation of mTOR in AD impairs the proteostasis network and inhibits autophagy, contributing to the accumulation of plaques and tangles 64 .The autophagy receptor p62 is a multifunctional protein, also known as sequestosome 1 (SQSTM1), that has been implicated in the pathology of AD due to its ability to bind to neurofibrillary tangles and thereby prepare it for degradation [65][66][67] .We found that PR significantly reduced p62 expression in female 3xTg mice, suggesting that PR activates autophagy in 3xTg mice (Fig. 7D).A similar trend which did not reach statistical significance was observed in male 3xTg mice (Fig. S4D).These data are consistent with a model in which PR inhibits mTORC1 signaling and thereby activates autophagy, reducing AD pathology (Fig. 7E).

PR rescues hippocampal-dependent spatial learning associated memory deficits
We studied the effects of PR on cognition by performing behavior assays on 12-monthold mice, which had been fed the indicated diets for 6 months.We conducted an Open Field Test (OFT), tested Novel Object Recognition (NOR), and examined performance in a Barnes Maze (BM).In the OFT, we observed an overall effect of genotype in females, with 12-month-old female 3xTg mice, those on a PR diet, showing increased exploratory behavior (Fig. S5A).NOR tests the preference for exploring a familiar object vs. a new object and are quantified based on a discrimination index (DI) following a short-term memory (STM) test and a long-term memory test (LTM); A positive DI implies a preference for exploring novelty, indicating that the familiar object's memory persists and the mice favor exploring the new object.In agreement with the literature 68 , we found that the 3xTg female mice showed more preference towards familiar object than the displaced object and thereby had impaired NOR (decreased DI) during both STM and LTM tests; PR-fed 3xTg females had significantly improved performance on NOR relative to the 3xTg controlfed females, while the performance of NTg mice was not affected by PR (Fig. 8A).Finally, we determined if PR could improve deficits in spatial learning and memory using BM; in this assay, the mice were required to locate an escape box placed at the target hole using spatial cues during the acquisition phase.On day 5 and 12 of the trial, a spatial working memory test (STM and LTM) was conducted to evaluate the reference memory of the previously learned target hole.PR-fed females -regardless of genotype -located the escape box more quickly during the training phase; when testing STM, we found that PR-fed 3xTg females found the escape box much more rapidly than 3xTg females fed the control diet.(Fig. 8B).
Male 3xTg mice on a Control diet travelled a shorter distance and displayed more anxietylike behaviors than Control-fed NTg males in the OFT.PR strongly increased both the distance covered and the velocity of 3xTg-fed male mice, without affecting the performance of NTg controls (Fig. S5B).3xTg male mice on a control diet had significantly impaired performance on a LTM during NOR, and this was significantly improved by PR (Fig. 8C).In the BM, we observed that Control-fed 3xTg males had impaired performance during both the short-term and long-term memory tests; in both cases, PR-feeding rescued this deficit in 3xTg males (Fig. 8D).To summarize, PR significantly improved memory deficits caused by AD in both males and females.Specifically, PR-fed females showed improved recognition memory, as observed in both shortterm and long-term memory tests.On the other hand, PR-fed males showed greater improvements in spatial memory.

PR improves the survival of 3xTg mice.
Throughout the course of all our experiments, we observed that 3xTg mice, especially males, had a propensity to die as they approached one year of age.We are not the first to make this observation; previous studies have observed a high mortality rate in male 3xTg-AD mice, varying from 33% 69 to 100% 70 depending upon age.Female 3xTg mice have a lower mortality rate than males 71 .In agreement with these findings, we observed that 3xTg males have a shorter lifespan than female mice (p=0.007,log-rank test stratified by sex) (Figs.9A-B).Furthermore, a PR diet increased the survival of both male and female 3xTg mice (p=0.03,log-rank test stratified by diet), and similar trends were observed when each sex was analyzed separately (Figs.9A-B).

Discussion
Dietary protein is a critical mediator of health in both rodents and in humans.In both rodents and in humans, PR improves metabolic health 15,19,20,22 ; and in mice, PR not only improves metabolic health, but extends lifespan, at least in males [23][24][25][26] .The geroscience hypothesis holds that interventions that slow aging should be effective in preventing or treating age-related diseases; and nowhere is the need for new treatment options more evident than in the case of Alzheimer's disease (AD).
Here, we examined the hypothesis that PR can prevent or slow the progression of AD using the 3xTg mouse model of this disease.We initiated treatment at 6 months of age -an age at which 3xTg mice show cognitive deficits as well as aspects of AD pathology, making this a reasonably translatable model, as treatment of humans with AD typically begins after AD related cognitive symptoms are evident.We have found that PR has significant benefits for AD neuropathology, cognitive performance, and overall survival; and our results suggest that PR may be an effective intervention in AD in both males and females.
The mechanisms by which PR improves the cognitive performance of 3xTg mice remains to be determined.We observed that PR significantly decreased levels of p-tau, Aβ plaques, and levels of Aβ 1-40 in female mice; in contrast, in males PR resulted in a statistically significant decrease only in levels of p-tau.Additionally, PR-fed female mice had decreased neuroinflammation as assessed by staining for astrocytes and microglia, while there was no effect of PR on neuroinflammation in males.Finally, neuroinflammation is a key pathological feature of AD, and we assessed activation of astrocytes and microglia via immunofluorescence.As both sexes showed similar cognitive benefits from PR, our data suggests the reduced tau phosphorylation may drive the benefits of PR for cognition in AD.There is similarly a strong association between phosphorylated tau and cognitive decline in humans 72 .
Epidemiological studies have clearly indicated that metabolic abnormalities can exacerbate AD pathology and cognitive impairment [73][74][75] .Our findings were largely consistent with previous research, which has shown that AD in the 3xTg mouse model induces a diabetic-like phenotype characterized by glucose intolerance, hyperinsulinemia, and hyperglycemia 52 ; intriguingly, these deficits were more pronounced in females.We find that PR effectively rescues the impaired glucose handling of 3xTg mice.It remains to be determined if the metabolic benefits of PR -which are more pronounced in females -contributes to the stronger effect of PR on AD pathology in 3xTg mice.
A number of studies have shown that mTORC1 activity is increased in the brains of mice and humans with AD, and previous studies have demonstrated that mTORC1 activity is positively correlated with tau phosphorylation 37,76 , suggesting a role for increased mTORC1 signaling in the etiology of AD.Hyper activation of mTORC1 signaling suppresses autophagy, which can contribute to the accumulation of plaque deposits and tau tangle formation, both hallmarks of AD 62,77   .In the present study, we have found that mTORC1 activity, as assessed by phosphorylation of mTORC1 substrates, is increased in the brains of 3xTg mice.PR decreased this elevated mTORC1 activity, and as noted above, decreased tau phosphorylation; we also observed an increase in autophagy as assessed by levels of p62.In combination, our results suggest a mechanistic model in which PR protects mice from worsening AD pathology and preserves cognition via inhibition of brain mTORC1 activity, which reduces AD pathology -especially tau phosphorylation -by increasing autophagy.While proving this model will take additional research, we were surprised to note that inhibition of mTORC1 activity by PR is not sufficient to significantly reduce Aβ plaques, the levels of Aβ 1-40, or affect neuroinflammation in male mice.The lack of significant impact on Aβ plaques and the levels of Aβ 1-40 suggests that mTORC1 may not be the sole driver of the effects of PR on AD pathology, implicating the role of other factors and pathways in accumulation of plaque deposits.
We further characterized PR-induced changes at the molecular level using a targeted metabolomics and lipidomics-based approach on blood plasma and brain respectively.
Intriguingly, and in agreement with our prior work on PR in the liver, we found that the molecular response to PR was sex-specific, with essentially no overlap in the molecular pathways engaged by PR in 3xTg males and females.In 3xTg females, we observed significant changes in methionine metabolism and glycine/serine/threonine metabolic pathways, which were significantly altered in the plasma.Cysteine and methionine are sulfur containing amino acids that play an important role in the production of glutathione, a potent antioxidant that is decreased in the brains of AD patients 78,79 .Similarly, alterations in glycine, serine, and threonine metabolism have been linked to AD and cognitive deficits as well as to longevity [80][81][82] .There was no overlap in the response to PR between male and female 3xTg mice; we observed upregulation of arginine/proline biosynthesis and aminoacyl-tRNA synthesis in 3xTg male mice fed a PR diet.
Arginine is the substrate for nitric oxide synthase (NOS), which plays a role in redox stress in the brain and which is associated with AD pathology 83 .Intriguingly, although there was no overlap between pathways altered by PR in male and female 3xTg mice, we did identify overlapping pathways in the non-transgenic controls, suggesting that AD may exacerbate sex differences.
In both sexes, we observed changes induced by PR that may potentially contribute to the cognitive benefits of this diet.3xTg females fed a PR diet showed decreased levels of plasma GTP, and purine nucleotide synthesis has been previously linked to neuroinflammation and cognitive decline in AD 84 .In addition, we observed increased levels of histidine and ornithine, which are involved in the synthesis of neurotransmitters such as histamine and GABA, respectively.Levels of both histamine and GABA are decreased in the plasma of AD patients 85- 87 .3xTg males fed a PR diet had decreased levels of proline and arginine, which have been identified as biomarkers for AD progression 88,89 .Both amino acids serve as precursors for the synthesis of glutamate and studies have suggested that alterations in the arginine and proline metabolism pathways can lead to dysregulation of glutamate levels, which can contribute to neurodegeneration in AD 90 , while arginase is upregulated in AD brains 90,91 .Of course, these are only correlations, and significant future work will be needed to examine if these changes in metabolite levels are causal for the cognitive benefits of PR.
We initially performed untargeted lipidomics on the hippocampus and cortex of 3xTg mice.Interestingly, while the lipid profiles were strongly grouped by brain region, there was almost no overall effect of diet on lipid profiles in either hippocampus or cortex.We observed that the levels of several plasmalogens (Ether PE), and sphingolipids were altered by PR in both sexes especially in the cortex.Plasmalogens, also known as ether lipids, are membrane lipids that have a vinyl-ether capable of scavenging reactive oxygen species which protects other phospholipids from lipid peroxidation, which is associated with autophagy, cellular dysfunction, and increased membrane permeability 92 .Plasmalogens are increased in AD, and the reduced levels of specific EtherPEs could be associated with decreased oxidative stress.There were also PR-induced changes in Ether lipids, which are strongly correlated with oxidative stress in AD. 55 .
Several sphingolipid species, including ceramides, glucosylceramides, and gangliosides, have been found to play a role in the pathogenesis of AD [93][94][95] .To explore this further, we conducted targeted lipidomics on the whole brains of NTg and 3xTg mice fed either a control diet or a PR diet.Interestingly, in 3xTg female mice on the PR diet, we observed a downregulated pattern of many subclasses of ceramides, sphingomyelins, and glucosylceramides.This reduction in sphingolipid levels in females could be attributed to the potential consequences of reduced plaques, as sphingolipids are known to be involved in Aβ processing and aggregation 61 .In contrast, we did not observe significant changes in sphingolipid levels in male mice under the same conditions.Finally, we observed that 3xTg mice of both sexes, but particularly males fed a Control diet, had reduced survival.Previous studies on 3xTg mice, which develop neuropathological symptoms associated with AD, have reported sex differences in the progression of these symptoms, as well as differences in morbidity and mortality rates 69,[96][97][98] .Specifically, male 3xTg mice have been shown to have increased mortality 99 due to impaired neuroimmune system function compared to females.Here, we observed an overall negative effect of male sex on the survival of 3xTg mice, and an overall positive effect of PR on 3xTg survival.Interestingly in a previous study we did not observe a positive effect of PR on the lifespan of wild-type female mice 23 , and it would be interesting to know if the effect on female survival we observed here is a result of AD, genetic background, or age of diet start.
Limitations of the present study include the exclusive use of the 3xTg mouse model; the use of other AD mouse models could give improved insight, particularly into understanding if the effects of PR are mediated by its effects on Aβ, tau, or both.Further, as different strains of mice have different metabolic responses to PR, the effects of PR on AD development and progression may vary as a result of genetic background.Our molecular analyses were relatively targeted concentrating on plasma and brain tissues, as well as probing the mTOR signaling pathways.
However, to gain a comprehensive understanding, more extensive research will be necessary to elucidate the intricate molecular mechanisms engaged by PR within the brain.Finally, our studies of male mice in particular were impacted by the reduced survival of 3xTg males, which limited the sample size of animals available for analysis of AD pathology.
In conclusion, we have shown that protein restriction can protect 3xTg AD mice from multiple aspects of the disease, including disrupted glucose homeostasis, development of AD pathology including Aβ plaques and phosphorylated tau, the development of cognitive deficits, and even increase survival.We started this dietary intervention at 6 months of age, which while fairly young for a mouse -roughly equivalent to a human in their 30's -is subsequent to the beginning of AD pathology and cognitive deficits in this model.Thus, our work suggests that PR can be deployed after the disease is symptomatic.Additional research will be required to determine if there are potentially negative effects of PR, particularly regarding effects on muscle mass and strength in older adults, which may limit the translatability of PR diets to humans.Finally, our results in wild-type mice suggest that protein quality -the specific amino acid composition of the dietary protein -has important effects on metabolic health as well as longevity 23,26,100   .Hence, we will continue to determine if individual amino acids contribute to the beneficial effects of PR on AD pathology and cognition in our future studies.Our results support an emerging model that geroprotective interventions may be of use in the treatment of AD, and suggest that PR, or pharmaceutical or dietary regimens that engage these same molecular mechanisms, may be a new and effective way to prevent or delay the progression of this age-related disease.

Animals
All procedures were performed in accordance with institutional guidelines and were approved by the Institutional Animal Care and Use Committee of the William S. Middleton Memorial Veterans Hospital (Madison, WI, USA).Male and female homozygous 3xTg-AD mice and their non-transgenic littermates were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and were bred and maintained at the vivarium with food and water available ad libitum.
Prior to the start of the experiments at 6 months they were randomly assigned to different groups based on their body weight, diet and genotype.Mice were acclimatized on a chow diet (Purina 5001) for one week before experiment start and were housed 2-3 per cage.All mice were maintained at a temperature of approximately 22°C, and health checks were completed on all mice daily.At the start of the experiment, mice were randomized to receive either a 21% protein diet (Control, TD.180161) or a 7% protein diet (PR, TD.10192) obtained from Envigo.Full diet descriptions, compositions and item numbers are provided in Table S1.

In vivo Procedures
Glucose and pyruvate tolerance tests were performed by fasting the mice overnight for 16 hours and then injecting glucose (1 g kg −1 ) or pyruvate (2 g kg -1 ) intraperitoneally (i.p.) as previously described 101,102 .For insulin tolerance we fasted the mice for 4 hours and injected insulin intraperitoneally (0.75 U kg 1 ).Glucose measurements were taken using a Bayer Contour blood glucose meter (Bayer, Leverkusen, Germany) and test strips.Mouse body composition was determined using an EchoMRI Body Composition Analyzer (EchoMRI, Houston, TX, USA).For determining metabolic parameters [O2, CO2, food consumption, respiratory exchange ratio (RER), energy expenditure] and activity tracking, the mice were acclimated to housing in a Oxymax/CLAMS-HC metabolic chamber system (Columbus Instruments) for ∼24 h and data from a continuous 24 h period was then recorded and analyzed.Mice were euthanized by cervical dislocation after a 3 hr fast and tissues for molecular analysis were flash-frozen in liquid nitrogen or fixed and prepared as described in the methods below.

Behavioral assays
All mice underwent behavioral phenotyping when they were twelve months old.The Novel object recognition test (NOR) was performed in an open field where the movements of the mouse were recorded via a camera that is mounted above the field.Before each test mice were acclimatized in the behavioral room for 30 minutes and were given a 5 min habituation trial with no objects on the field.This was followed by test phases that consisted of two trials that are 24 hrs apart: Short term memory test (STM and Long term memory test (LTM).In the first trial, the mice were allowed to explore two identical objects placed diagonally on opposite corners of the field for 5 minutes.Following an hour after the acquisition phase, STM was performed and 24 hrs later, LTM was done by replacing one of the identical objects with a novel object.The results were quantified using a discrimination index (DI), representing the duration of exploration for the novel object compared to the old object.
For Barnes maze, the test involves 3 phases: habituation, acquisition training and the memory test.During habituation, mice were placed in the arena and allowed to freely explore the escape hole, escape box, and the adjacent area for 2 min.Following that during acquisition training the mice were given 180s to find the escape hole, and if they failed to enter the escape box within that time, they were led to the escape hole.After 4 days of training, on the 5 th day the mice were given 90s memory probe trials.The latency to enter the escape hole, distance traveled, and average speed were analyzed using Ethovision XT (Noldus).

Immunoblotting
Tissue samples from brain were lysed in cold RIPA buffer supplemented with phosphatase inhibitor and protease inhibitor cocktail tablets (Thermo Fisher Scientific, Waltham, MA, USA) using a FastPrep 24 (M.P. Biomedicals, Santa Ana, CA, USA) with bead-beating tubes (16466-042) from (VWR, Radnor, PA, USA) and zirconium ceramic oxide bulk beads (15340159) from (Thermo Fisher Scientific, Waltham, MA, USA).Protein lysates were then centrifuged at 13,300 rpm for 10 min and the supernatant was collected.Protein concentration was determined by Bradford (Pierce Biotechnology, Waltham, MA, USA).20 μg protein was separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) on 8%, 10%, or 16% resolving gels (ThermoFisher Scientific, Waltham, MA, USA) and transferred to PVDF membrane (EMD Millipore, Burlington, MA, USA).The phosphorylation status of mTORC1 substrates including S6K1 T389 and 4E-BP1 T37/S46 were assessed in the brain along with p62 protein receptor.Tau pathology was assessed by western blot with anti-tau antibody.Antibody vendors, catalog numbers and the dilution used is provided in Table S11.Imaging was performed using a GE ImageQuant LAS 4000 imaging station (GE Healthcare, Chicago, IL, USA).Quantification was performed by densitometry using NIH ImageJ software.

Histology for AD neuropathology markers:
Mice were euthanized by cervical dislocation after a 3 hour fast, and the right hemisphere was fixed in formalin for histology whereas the left hemisphere was snap-frozen for biochemical analysis.Formalin fixed brain sections were analyzed for plaques using thioflavin S staining.
Briefly deparaffinized and rehydrated slides were incubated for 10min in 1% thioflavin-S (Sigma; #T3516) which was dissolved in 50% ethanol and the slides were rinsed in 80% ethanol and 50% ethanol and mounted with aqueous mounting media with DAPI.For astrocytic and microglial activation, Brains were analyzed with anti-GFAP, and anti-Iba1 antibodies respectively.The following primary antibodies were used: anti-GFAP (ThermoFisher; # PIMA512023; 1:1,000), anti-IBA1 (Abcam; #ab178847; 1:1,000).Sections were imaged using an EVOS microscope (ThermoFisher Scientific Inc., Waltham, MA, USA) at a magnification of 4X and 10X.Image-J was used for the quantification and analysis of plaques, thioflavin-S images were converted into binary images via an intensity threshold and using particle analyzer plaques were counted. 103.

Metabolite extraction
Plasma (20µL of) was transferred to an individual 1. Next, the pooled metabolite extract for each sample was transferred to a 1.5 mL microcentrifuge Eppendorf tube and completely dried using a Thermo Fisher Savant ISS110 SpeedVac.Dried metabolite extracts were resuspended in 100 µL of LCMS-graded water following microcentrifugation for 5 minutes at 21,000 xg at 4C to pellet any remaining insoluble debris.Supernatant was then transferred to a glass vial for LC-MS analysis.

Metabolomics analysis:
The metabolites were initially normalized using a log base 2 transformation.Additionally, to control for false positives, P values were adjusted using the Benjamini-Hochberg procedure with a false discovery rate (FDR) of 20%.Subsequently, pathway analysis was conducted using the online tool, MetaboAnalyst(https://docs.polly.elucidata.io/Apps/Metabolomic%20Data/El-MAVEN.html).
The Pathway Analysis function of the tool was employed by inputting a list of significantly altered metabolites based on their corresponding human metabolome database (HMDB) IDs obtained from the linear model with a significance threshold of p<0.05.

Untargeted Lipidomics
All solvents used for lipid extraction were LCMS grade or better.MeOH and ethyl acetate (EtOAc) were purchased from Honeywell (LC230-4, 34972-1L).Isopropanol (IPA) was purchased from Fisher (A461-4), and water was purchased from ThermoFisher (600-30-78).Lipids were extracted from 20 mg brain tissue.Extractions were done on ice using solvents chilled to 4 °C.10 µL SPLASH mix, 30 pmol d7 Ceramide, and 10 pmol d7 PG per sample were used as internal standards.A process blank containing extraction solvent only was extracted with the samples.
Lipids were resuspended in 150 µL 100% MeOH for mass spectrometry analysis.At this point, an insoluble precipitate was observed in all samples.Samples were stored at -20°C for up to 2 months before analysis.
For untargeted lipidomics in positive mode, lipids were diluted at 1:30 in MeOH.Samples were centrifuged before dilution to avoid injecting the insoluble precipitate.Negative mode samples were not diluted.3 µL of each diluted sample was injected in positive mode, and 5 µL was injected in negative mode.Lipids were separated using an Acquity BEH C18 column (Waters 186009453, 1.7 µm 2.1 x 100 mm) at 50 °C with a VanGuard BEH C18 precolumn (Waters 18003975) on an Agilent 1260 Infinity II UHPLC system.The chromatographic gradient began at 85% mobile phase A, which consisted of 60:40 ACN:H2O with 10 mM ammonium formate and 0.1% formic acid, and 15% mobile phase B consisted of 9:1:90 ACN:H2O:IPA with 10 mM ammonium formate and 0.1% formic acid.The flow rate was 0.5 mL/min.The gradient increased to 30% mobile phase B during the next 2.4 minutes.The gradient then increased to 48% until 3 min, and then to 82% at 13.2 min.From 13.2-13.8min, the gradient increased to 99%, and stayed at 99% until 16 min.At 16 min, re-equilibration to 15% mobile phase B began and was held until 20 min.
In negative mode, the gas temperature was maintained at 250°C at a flow rate of 12 L/min.The sheath gas was maintained at 375 °C at a flow rate of 12 L/min.The nebulizer was set to 30 PSI.Vcap was set to 4000 V, the skimmer was set to 75 V, the fragmentor was set to 190 V, and the octapole radiofrequency peak was set to 750 V.In positive mode, all mass spec settings were the same, except that the nebulizer was set to 35 PSI, and the sheath gas was maintained at 300 °C with a flow rate of 11 L/min.Reference masses used for positive mode were 121.05 and 922.01 m/z, and reference masses used for negative mode were 112.98 and 1033.99 m/z.For both ionization modes, the acquisition rate was 3 spectra/second, and the m/z range was 100-1700 m/z.For MS2 scans in both modes, the isolation width was set to narrow (1.3 m/z), the acquisition rate was 2 spectra/second, and the collision energy was fixed at 25 V. Precursors were excluded after 1 spectrum.Data files were collected in.d format.LipidAnnotator was used to identify lipids from MS/MS data from pooled samples.Identified lipids were exported in PCDL format to create compound libraries for each gender.Agilent Profinder was used to identify compounds from the libraries in the MS1 data for each sample.Identified compounds and intensities for each sample were exported as .csvfiles, and compound intensities were normalized to internal standards using an in-house R script.Samples were excluded from statistical analysis if lipid concentrations were 2 or more standard deviations from the mean.
Targeted analysis was performed in positive ionization mode on an Agilent 1290 Infinity II UHPLC coupled to an Agilent 6495C triple quadrupole MS.Extracts were separated using an Acquity BEH C18 column (Waters 186009453, 1.7 µm 2.1 x 100 mm) connected to a VanGuard BEH C18 precolumn (Waters 18003975) at 60 °C.Extracts were diluted 1:30 in IPA prior to injection.Mobile phases for the separation gradient were of the same composition as in the untargeted lipidomic analysis.For targreted sphingolipid separation, the gradient began with 30% B and increased to 60% over 1.8 min, then increased to 80% until 7 min and 99% until 7.14 min, which was maintained until 10 min.Sphingolipids were quantified using dynamic reaction monitoring (dMRM).The gas temperature was maintained at 210 °C and 11 L/min while the sheath gas temperature was 400 °C and 11 L/min flow.The capillary voltage was 4000 V and nozzle voltage was 500 V. Nebulizer pressure was 30 PSI.Low-pressure RF was 120 and highpressure was 190.Retention time windows and collision energies were optimized based on internal standards of the same sphingolipid species.Data was processed in the Agilent MassHunter Wokstation, and sphingolipids were quantified by peak height based on the relative concentration of the appropriate internal standard within the samples.

Assay kits
The quantification of amyloid-beta 40 (Aβ40) in the brain was performed using the enzyme-linked immunosorbent assay (ELISA) technique.The Human Aβ40 ELISA kit (Invitrogen, USA, cat# KHB3482) was utilized in accordance with the manufacturer's instructions.

Statistical Analysis
All statistical analyses were conducted using Prism, version 9 (GraphPad Software Inc., San Diego, CA, USA).Tests involving multiple factors were analyzed by either a two-way analysis of variance (ANOVA) with diet and genotype as variables or by one-way ANOVA, followed by a Dunnett's, Tukey-Kramer, or Sidak's post-hoc test as specified in the figure legends.Survival analyses were conducted in R using the "survival" package (Therneau, 2015).Kaplan-Meir survival analysis of 3xTg mice was performed with log-rank comparisons stratified by sex and diet.Cox proportional hazards analysis of 3xTg mice was performed using sex and diet as covariates.Alpha was set at 5% (p < .05considered to be significant).Data are presented as the mean ± SEM unless otherwise specified.
5 mL microcentrifuge tube and incubated with 400µL -80°C 80:20 Methanol (MeOH):H2O extraction solvent on dry ice for 5 minutes post-vortexing.Serum homogenate was centrifuged at 21,000 xg for 5 minutes at 4°C.Supernatant was transferred to 1.5 mL microcentrifuge tube after which the remining pellet was resuspended in 400 µL -20°C 40:40:20 Acetonitrile (ACN):MeOH:H2O extraction solvent and incubated on ice for 5minutes.Serum homogenate was again centrifuged at 21,000 xg for 5 minutes at 4°C after which the supernatant was pooled with the previously isolated metabolite fraction.The 40:20:20 ACN:MeOH:H2O extraction was then repeated as previously described.

Figure 1 :Figure 3 :
Figure 1: Protein restriction prevents weight and fat mass gain in 6-month-old 3xTg mice and NTg controls of both sexes.

Figure 4 :
Figure 4: PR induces sex-specific shifts in the plasma metabolome of 3xTg mice.
Figure 5 A